CN107898524A - A kind of new magnetic particle gathers the construction method of cerebral ischemia animal model - Google Patents
A kind of new magnetic particle gathers the construction method of cerebral ischemia animal model Download PDFInfo
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- CN107898524A CN107898524A CN201711426136.9A CN201711426136A CN107898524A CN 107898524 A CN107898524 A CN 107898524A CN 201711426136 A CN201711426136 A CN 201711426136A CN 107898524 A CN107898524 A CN 107898524A
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- animal model
- cerebral ischemia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D1/00—Surgical instruments for veterinary use
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
Abstract
The invention discloses the construction method that a kind of new magnetic particle gathers cerebral ischemia animal model, by injecting the buffer solution being configured to containing magnetic nano particle in living animal, and by surgical operation means it is to wrap soft wire magnetic stripe in internal carotid, so that living animal is wrapping formation thrombus 2h at soft wire magnetic stripe, its cerebral ischemia animal model built can be identical the symptom of cerebral ischemia, smaller to animal model damage, the death rate of its animal model structure is relatively low.
Description
Technical field
The present invention relates to animal pharmacological experiment technical field, a kind of more particularly to new magnetic particle gathers brain and lacks
The construction method of blood animal model.
Background technology
Transient ischemic attack (TTA) is that transience blood supply deficiency occurs for arteria carotis or Vertebro-basilar System,
Focal cerebral ischemia is caused to cause burst, transience, invertibity neurological dysfunction.Break out last for several minutes, usually 30
Recover completely in minute, it is small more than 2 to leave slight neurologic impairment performance, or CT and MRI display brain tissue ischemia signs often
As.TIA is apt to occur in 34~65 years old, and over-65s account for 25.3%, and male is more than women.Morbidity is unexpected, more in Body Position Change, activity
Excessively, neck is fallen ill when rotating or bend and stretch suddenly.Morbidity absence of aura, there is transient focal neurological signs.One
As unconscious obstacle, last 5~20 minutes, can recurrent exerbation, but it is interior generally when 24 is small recover completely, no sequel, in neck
The most common symptoms of TIA of arterial system are monoplegia, hemiplegia, hemidysesthesia, aphasia, monocular vision obstacle etc., can also be gone out
Now with tropism hemianopsia etc..Main performance：It is simple eye blackout, or visual loss occur suddenly, or white flicker, or the visual field lacks
Damage, or diplopia, last for several minutes can recover.Contralateral limbs are slight hemiplegia or hemiparesthesia.Dominant hemisphere is damaged appearance one
The aphasia or appraxia or alexia or logagraphia for the property crossed, or facial muscle, lingualis are powerless at the same time.Occasionally there is homonymous hemianopsia.Its median ocellus goes out suddenly
Existing blackout is the characteristic symptom of internal carotid branch ophthalmic artery ischemic.Of short duration mental symptom and the disturbance of consciousness are even also
It can be seen that.
Research cerebral ischemia is carried out on being influenced caused by body by establishing cerebral ischemia animal model in clinical trial, still
In the prior art, anesthetized animal is used at present, and cerebral ischemia animal model is made oppressing or blocking by surgical means, its
Simulation to thrombosis does not conform to the actual conditions, while it may cause coup injury to blood vessel, and the death rate is higher, complicated,
Individual variation is big, its operation does not have invertibity, since transient cerebral ischemia has the possibility of multiple relapse, can not establish one
The cerebral ischemia animal model of sustainability.
The content of the invention
Gather cerebral ischemia in view of the deficiencies of the prior art, the present invention intends to providing a kind of new magnetic particle to move
The construction method of thing model, can be identical the symptom of cerebral ischemia, smaller to animal model damage, its animal model structure
The death rate is relatively low.
To achieve the above object, the present invention provides following technical solution：A kind of new magnetic particle gathers cerebral ischemia animal
The construction method of model, this method step is as follows,
(1) buffer solution being configured to magnetic nano particle is taken, by the buffer injection to living animal；
(2) animal model is weighed, anaesthetized；
(3) four limbs of animal model are fixed and performed the operation preparation；
(4) animal model is taken into 3cm neck median incisions, exposes in left common carotid, external carotid artery and neck move successively
Arteries and veins；
(5) soft wire magnetic stripe is wrapped in internal carotid；
(6) after ischemic 2h, anesthetized animal, unties and takes out linear magnetic stripe again, sutures tissue cut；
(7) hemoperfusion 24h is recovered.
As a further improvement on the present invention, magnetic-particle is enclosed with inert material.
As a further improvement on the present invention, the magnetic nanoparticle is the metal magnetic nano particle less than 150nm.
As a further improvement on the present invention, the inert material is polyethylene glycol, glucose or glucan.
As a further improvement on the present invention, the molecular weight of the polyethylene glycol is 1000-2000.
As a further improvement on the present invention, granular size of the magnetic-particle after inert material is wrapped up is 100-
200nm。
As a further improvement on the present invention, the magnetic-particle is 5-20mg/ml in the concentration of buffer solution.
As a further improvement on the present invention, the injection system of the buffer solution in the step 1 is：Carotid injection, root
Injection accumulated dose 80-150ug/g is calculated according to weight.
As a further improvement on the present invention, monitor the anus temperature of animal model in real time in modeling process, keep rat
Anus temperature is at 36.5-37.5 DEG C.
Beneficial effects of the present invention are：By wrapping up soft linear magnet on the arteria carotis of animal model, and irrigate and contain
Be magnetic the buffer solution of nano particle, and the buffer solution containing magnetic nanoparticle circulates in vivo, by placing magnet bar
Embolism is formed during arteria carotis position, and the degree of embolism can be set by adjusting the magnetism of magnetic stripe, the Holistic modeling time is short,
Direct losses of traditional cerebral ischemic model to blood vessel are avoided, it is low to the extent of injury of animal model, improve building for animal model
Mould success rate, and the physical principle that its embolism is formed is approached with being actually formed the physical principle of embolism, can preferably simulate work
Influence of the body cerebral vessels embolism to body.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
1. experiment material：
1.1 animal material
SD rats are selected, male, wean mouse (birth the 21st day), buys from Medical Center of Fudan University's animal center.Animal
After buying back, random packet, sub-cage rearing.Keep quite during raising comfortable environment, allow its free water and feed.Experiment is opened
Two days experimenters before beginning respectively stroke, grasp rat once daily morning and afternoon, and allow rat to adapt to experimental provision and device used, with
Ask exclude as far as possible experimental situation caused by rat stress stimulation, ensure the accuracy of experimental result.
When 120 ± 10 days mouse ages are arrived in raising, the rat of 230 ± 30g of weight is selected.
1.2 main agents
75% alcohol, Iodophor, 10% chloraldurate (or yellow Jackets), 1% heparin, triphenyltetrazolium chloride
(triphenylte razoliumchloride, TTC), PBS；
1.3 key instrument
Rat plate, wiring (cotton rope), soft wire magnetic stripe, bulk pruning knife, scalpel, haemostatic clamp, eye scissors, the curved tweezer of ophthalmology,
Clipper for surgical use, surgical forceps, glass minute hand, medical dry cotton ball or gauze piece, 5ml syringes, alcolhol burner, paraffin, beaker, plate, hair
Fine droplets pipe, razor blade, angular pin, kidney basin, Self-made hook, emgloves, mitt and skin hand
1.4 laboratory apparatus
Slicer：LEICA CM 1800.
Constant temperature blanket：ALC-HTP
Constant Temp. Oven：JKDP-1 types.
Electronic balance：TIANFU DT-200A.
Microscope and supporting digital camera：OLMPUS IX71.
2. method：
The making of 2.1 soft linear magnetic stripes
The commercially available soft magnetic stripes of diameter 0.28mm, accurately cut 6cm, and after seeing the concordant blunt circle of the broken ends of fractured bone under mirror, a mark is done per 2cm
Note, puts 75% alcohol immersion 10min, and it is in ball-type that head end, which dips the paraffin that heat is melted to make its smooth, and it is spare to put the immersion of 1% heparin.
The preparation of 2.2 buffer solutions containing magnetic nanoparticle
Selecting Fe, Co, Ni magnetic nanoparticle, either ferroferric oxide magnetic nanoparticle or di-iron trioxide are magnetic
Nano particle, preferably selects diameter to be wrapped up in for the metal magnetic nano particle less than 150, its metal magnetic nano particle outer layer
Inert material is covered with, polyethylene glycol, glucose or glucan can be used, it is preferable to use molecular weight as the poly- of 1000-2000
Ethylene glycol, the granular size for wrapping the metal magnetic nano particle after inert material is 100-200nm, puts the immersion of 75% alcohol
After 10min, normal saline flushing, and by, into 5-20mg/ml, being preferably configured to 10mg/ml with physiological saline proportioning, configuring
Good buffer solution, its particle of magnetic nanoparticle is uniform, well dispersed in buffer solution.
2.3 modellings of the present invention
(1) rat body weight is weighed, magnetic nanoparticle is contained to the above-mentioned configuration of its carotid injection according to rat body weight
Buffer solution 80-150ug/g, jugular vein or tail vein injection can also be selected if necessary；
(2) dosage (3ml/kg) according to needed for weight calculates anesthesia, prepares 10% chloral hydrate anesthesia medicine, administering mode
Intraperitoneal injection of anesthesia is taken, treats that absent corneal reflex and anesthesia are abundant, takes back floating position to be fixed on mouse plate rat, four limbs make
Softly fixed with medical proof fabric.Sterile cleaning cotton ball is taken to wipe oral cavity and remove respiratory secretions；
(3) is placed in bicker side with the tongue of tissue clamps pull-out rat, to prevent glossoptosis from influencing rats breathing.Hand
Art operating area shaving preserved skin, iodophor disinfection, take 3cm neck median incisions, successively blunt separation neck subcutaneous tissue, according to solution
Order exposure left common carotid (CCA), external carotid artery (ECA) and internal carotid (ICA) are cutd open, operative incision is fixed with drag hook.
Give away one's position blood vessel when, gimmick is softly careful, avoid around its hetero-organization, blood vessel, nerve and thyroid gland, the damage of parathyroid gland
Wound.
(4) soft wire magnetic stripe ring-type is wrapped internal carotid ICA by, to be wrapped close to blood vessel, does not cause compressing to be advisable.
The control modeling time completes in 20 minutes.
(5) ischemics 2h, then slightly unties linear magnetic stripe with 10% chloraldurate intraperitoneal injection of anesthesia animal again, delays
Slow soft taking-up, then tissue cut is sutured again, iodophor disinfection, recovers hemoperfusion 24h, monitors its Brain circlulation.
During ischemic and recover in hemoperfusion 24h, all animals carry out anus temperature and monitor in real time, keep the anus of rat
Temperature is controlled to improve the survival rate of rat between 36.5-37.5 DEG C by constant temperature blanket or insulating box.
The preparation of 2.4 blank control groups
Other operations are the same, irrigate the buffer solution containing magnetic nanoparticle of equivalent, soft wire magnetic stripe is replaced with
Soft rubber bar.
The preparation of 2.5 line brush rat cerebral ischemia models
(1) prepared by lines bolt：Commercially available sub-thread diameter 0.28mm fishing lines, accurately cut 6cm, after seeing the concordant blunt circle of the broken ends of fractured bone under mirror,
A mark is done per 2cm, puts 75% alcohol immersion 10min, head end, which dips the paraffin that heat is melted and makes its smooth, to be in ball-type, put 1% heparin
Soak spare.
(2) surgical procedure
The chloraldurate (3ml/kg) of rats by intraperitoneal injection 10%, neck cropping disinfection after back of the body position is fixed, takes neck center to cut
Mouthful, blunt separation right common carotid artery (CCA), internal carotid (ICA) and external carotid artery (ECA) and hanging wire is spare, ligation ECA with
CCA, after closing ICA distal ends with artery clamp folder, rapidly in doing a kerf on CCA, is inserted into the above-mentioned fishing line got ready, line is inserted from notch
After entering ICA, the artery clamp that folder closes ICA is opened, soft line sending (dissection of crotch to arteria cerebri media) close at 18mm, there is resistance
Power is then stopped, fixing bolt line, is gently pulled out fishing line after 2h, is tightened ICA, skin suture.
It regard the modeling method of the present invention as experimental group one, the modeling method of line brush is experimental group two, experimental group one, reality
Test group two and blank group selects 12 rats respectively, the death rate of experimental group one is 91.7%, the death rate of experimental group two
For 50%, the death rate of blank group is 91.7%
3. neurological deficit scores
With reference to Longa and Bederson 5 points of preparation methods after Animal Anesthesia is regained consciousness 24h score.
0 point：Impassivity injury symptoms；
1 point：It is unable to full extension offside fore paw；
2 points：Turn-take to offside；
3 points：Topple over to offside；
4 points：Spontaneous it cannot walk, the loss of consciousness.
Score value is higher, illustrates that animal behavior obstacle is more serious.
The successful standard of model is：Body flexing to the left, the elongation of right side fore paw are better than left side, and tail bends towards left side；Carry tail
It is in mandatory figure when hanging.
Symptom 2-3 points of the death rate is high, and 1-2 points of the death rate is low, some rats second day or after a few days can be without obvious disease
Shape, but still have infarct after taking brain.
4. experimental result
With reference to 5 points of preparation methods of Longa and Bederson.Scoring 0 point (impassivity injury symptoms) can be considered that modeling fails, extremely
The rat died is equally considered as modeling failure, and the rat of this two class scoring is rejected, remaining score value animal includes experiment.
Experimental group one removes the rat of death, scoring of remaining 11 rats with reference to 5 points of preparation methods of Longa and Bederson
As a result it is respectively：1st, 1,1,2,1,3,2,1,1,1,1, model success rate is 83.3%,
Experimental group two removes the rat of death, scoring of remaining 6 rats with reference to 5 points of preparation methods of Longa and Bederson
As a result it is respectively：2nd, 4,2,3,2,4, model success rate is 25%,
Blank control group removes dead rat, remaining 11 rats commenting with reference to 5 points of preparation methods of Longa and Bederson
It is 0,0,0,0,0,0,0,0,0,0,0 to divide result, and model success rate is 0%.
It can show that the buffer solution containing magnetic-particle does not influence blood circulation by above experimental data, experimental group one
It is higher to model success rate, and models the scoring of successful animal model there was no significant difference, the modeling success rate of experimental group two
It is relatively low, and the scoring significant difference for modeling successful animal model is larger.
5. the application of cerebral ischemia animal model
Pass through the bulk damage degree of following Germicidal efficacy cerebral ischemia animal model：
TTC (2,3,5- triphenyltetrazolium chloride) is dyed：Broken end takes brain immediately after rat ischemia 2h Reperfu- sions 24h, soon
After quickly cooling is frozen, 5 brain pieces are cut altogether from the front to the back, thickness is about 2mm, is placed in 37 DEG C of incubation 30min in 2%T TC solution, sternly
Lattice lucifuge, shakes once every 5min, fixed in most 10% paraformaldehyde liquid of postposition, after by micro- sem observation, to observe bolt
The cerebral infarction volume of animal model after plug, has further more accurate and visual understanding to injury of the cerebral ischemia to body, can
Preferably it is applied in teaching experiment or new drug development drug study.
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment,
All technical solutions belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
For those of ordinary skill, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of new magnetic particle gathers the construction method of cerebral ischemia animal model, it is characterised in that this method step is as follows,
Step 1：The buffer solution being configured to magnetic nano particle is taken, by the buffer injection to living animal；
Step 2：By weighing living body of animal, anesthesia；
Step 3：The four limbs of living animal are fixed；
Step 4：Living animal is taken into 3cm neck median incisions, exposes in left common carotid, external carotid artery and neck move successively
Arteries and veins；
Step 5：Soft wire magnetic stripe is wrapped in internal carotid, causes ischemic；
Step 6：After ischemic 2h, anesthetized animal, unties and takes out linear magnetic stripe again, sutures tissue cut；
Step 7：Recover hemoperfusion 24h.
2. a kind of new magnetic particle according to claim 1 gathers the construction method of cerebral ischemia animal model, its feature exists
In：The magnetic-particle is wrapped with inert material.
3. a kind of new magnetic particle according to claim 1 gathers the construction method of cerebral ischemia animal model, its feature exists
In：The magnetic nanoparticle is the metal magnetic nano particle less than 150nm.
4. a kind of new magnetic particle according to claim 1 gathers the construction method of cerebral ischemia animal model, its feature exists
In：The inert material is polyethylene glycol, glucose or glucan.
5. a kind of new magnetic particle according to claim 4 gathers the construction method of cerebral ischemia animal model, its feature exists
In：The molecular weight of the polyethylene glycol is 1000-2000.
6. a kind of new magnetic particle according to claim 5 gathers the construction method of cerebral ischemia animal model, its feature exists
In：Granular size of the magnetic-particle after inert material is wrapped up is 100-200nm.
7. a kind of new magnetic particle according to claim 1 or 2 or 3 or 4 or 5 or 6 gathers the structure of cerebral ischemia animal model
Construction method, it is characterised in that：The magnetic-particle is 5-20mg/ml in the concentration of buffer solution.
8. a kind of new magnetic particle according to claim 7 gathers the construction method of cerebral ischemia animal model, its feature exists
In：The injection system of buffer solution in the step 1 is：Carotid injection, injection accumulated dose 80- is calculated according to weight
150ug/g。
9. a kind of new magnetic particle according to claim 1 gathers the construction method of cerebral ischemia animal model, its feature exists
In：Monitor the anus temperature of animal model in real time in modeling process, keep the anus temperature of rat at 36.5-37.5 DEG C.
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| CN201711426136.9A CN107898524A (en) | 2017-12-25 | 2017-12-25 | A kind of new magnetic particle gathers the construction method of cerebral ischemia animal model |
| CN201811591397.0A CN109549739B (en) | 2017-12-25 | 2018-12-25 | Experimental material for constructing magnetic particle-aggregated cerebral ischemia animal model |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109549739A (en) * | 2017-12-25 | 2019-04-02 | 温州医科大学附属第医院 | A kind of kit gathering cerebral ischemia animal model for constructing novel magnetic particle |
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| CN1660446A (en) * | 2004-12-28 | 2005-08-31 | 高焕民 | Method and application of producing disease model of local cerebral ischemia by using macaque |
| CN1330387C (en) * | 2005-02-07 | 2007-08-08 | 武汉理工大学 | Magnetic combine powder type vessel embolizing material |
| EP2496302B1 (en) * | 2009-11-02 | 2019-05-01 | Pulse Therapeutics, Inc. | Magnetomotive stator system for wireless control of magnetic rotors |
| CN103550834B (en) * | 2013-10-25 | 2015-07-15 | 北京大学 | Embolism material composition as well as preparation method and use thereof |
| GB201506381D0 (en) * | 2015-04-15 | 2015-05-27 | Isis Innovation | Embolization particle |
| CN108079366A (en) * | 2017-01-24 | 2018-05-29 | 珠海神平医疗科技有限公司 | A kind of liquid embolic material and preparation method thereof |
| CN107185054A (en) * | 2017-03-30 | 2017-09-22 | 向建平 | A kind of novel liquid embolic material and preparation method thereof |
| CN107898524A (en) * | 2017-12-25 | 2018-04-13 | 温州医科大学附属第医院 | A kind of new magnetic particle gathers the construction method of cerebral ischemia animal model |
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| CN109549739A (en) * | 2017-12-25 | 2019-04-02 | 温州医科大学附属第医院 | A kind of kit gathering cerebral ischemia animal model for constructing novel magnetic particle |
| CN109549739B (en) * | 2017-12-25 | 2021-04-02 | 温州医科大学附属第一医院 | Experimental material for constructing magnetic particle-aggregated cerebral ischemia animal model |
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Application publication date: 20180413 |